Gene. 120 (1992) 291-295 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

291

0378-l 119/92/%05.00

06698

Cloning, expression and tissue distribution fibroblast growth factor receptor subtype 4

of the

gene

encoding

rat

(Intracellular receptor; lung cDNA; molecular sequence data; polymerase chain reaction; protein-tyrosine kinase)

Robert A. Horlick,

Sylvia L. Stack and Gary M. Cooke

The Du Pont Merck Pharmaceutical Co., P.O. Box 80400. Experimental Station, Wilmington, DE 19880-0400. USA. Tel. (302) 695-7247 Received

by W.P. Sisk: 30 March

1992; Revised/Accepted:

7 April/25

April 1992; Received

at publishers:

26 June 1992

SUMMARY

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFRI) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.

INTRODUCTION

A family of receptors that binds acidic and basic fibroblast growth factor (aFGF and bFGF) and at least some members of the related family of FGF proto-oncogenes have recently been identified. The four receptor subtypes within this family that have been cloned and characterized include: FGFRl or JEg (Lee et al., 1989; Pasquale and Singer, 1989; Isacchi et al., 1990), FGFR2 or bek (Dionne et al., 1990; Houssaint et al., 1990), FGFR3 and cek2 (Pasquale, 1990; Keegan et al., 1991), and FGFR4 (Par&men

Correspondence to: Dr. R.A. Horlick, The DuPont

Merck

Pharmaceutical

USA. Tel. (302) 695-7247; Abbreviations:

E400/5225,

aa, amino acid(s); aFGF,

fibroblast growth factor; FGFR, FGF FGFR; h-, human; Ig, immunoglobulin;

acidic FGF; bFGF,

receptor; FGFR, gene encoding kb, kilobase or 1000 bp; nt,

oligo, oligodeoxyribonucleotide;

sulfate; TyrK, tyrosine

basic FGF;

to RNA; A, deletion; FGF,

ORF, open reading frame;

PCR, polymerase chain reaction; r-, rat; RACE, rapid cDNA ends; SSC, 0.15 M NaCl/O.OlS M Na,.citrate sodium dodecyl

Station,

DE 19880-0400,

Fax (302) 695-7054.

bp, base pair(s); cDNA, DNA complementary

nucleotide(s);

Experimental

Co., Wilmington,

kinase.

amplification of pH 7.6; SDS,

et al., 1991). These receptors belong to the protein TyrK as well as to the Ig-like superfamilies of hormone receptors. Members of this family characteristically contain a hydrophobic signal sequence, an acidic region, two or three Iglike domains, a single hydrophobic transmembrane domain, and a cytoplasmically located ‘split’ TyrK enzymatic moiety (Fig. 1). The mRNAs encoding FGFRl and FGFR2 are also known to include several alternatively spliced variants. In one alternatively spliced form, the most N-terminal of the three Ig-like domains is absent (Johnson et al., 1990; Mansukhani et al., 1990). Furthermore, there are known to be three alternative exons that can be used to encode the C-terminal half of the third Ig domain (Johnson et al., 1991). The choice of exon utilization in the third Ig domain can determine the ligand-binding specificity of the resulting receptor (Miki et al., 1992; Werner et al., 1992) and whether the receptor is anchored to the cell membrane or is secreted from the cell (Johnson et al., 1990). Finally, Hou et al. (1991) have reported a variant of FGFRl that they term the ‘ y N-terminal motif which contains no signal sequence or acidic box. We report here the cloning and characterization of the rat homologue of the FGFR4 receptor that, like the y N-terminal motif of FGFRl, lacks a signal sequence and acidic box.

292 -60

-10 .

. CATCAATAACGAGGACCCUCCTCAGCAGCTCCTCG t60 . . . . CGGGAAU\CTGmAAATTCCTGTCCAGCTGCAGGGAAC GNTVKFRCPAAGNPMPTIHWLKNGQAFHGENRIGGIRLRH T flS0 . . CCAACACTGGAGCCTCGTGATGGAGAGCGTGGTGCCCTCAG QHW S LV MESVVP S D R

.

.

t10 . . GCBTGGAUlAGAAACTGCACGCGGTACCTGC MEKKLHAVPA t120 .

.

.

D

R

+240 .

. GT

Y

TCLV

ENS A

L V

GS

. I

RY

S N

Y

LL

DVL

. . GTCCCCGCACCGGCCCATCCTGCAGGCGGWLCTCCCAGCCT SPHRPILQAGLPANTTAVVGSNVELLCKVYSDAQPHIQWLl30

D +4ao +420 . . . . . . . GAAGCACATCGTTATCAACGGCAGCAGTCTCGGCGCTGAT~TTTCCCCTACGTA~GTCCT~GA~~~~T~TA~T~~ffiT~~T~TGTATCT~~ffiTGTC KHIVINGSSLGADGFPYVQVLKTTDINSSEVEVLYLRNVS170 A P V +600 t540 . . . . . . . GGCTWLGGATGULCX;GGAGTACACCT~G~CTC~TC~CTCTCCTAC~GT~~GT~T~CAGT~TACCC~~~~~~CCTC~GT~~~~~C AEDAGEYTCLAGNSIGLSYQSAWLTVLPAEEEDLAWTTAT210 A A P T t720 +660 . . . . . . ATCGGRGGCCAWLTATACTATTATCCTATATGTCCAT~T~CT~TTT~TTTT~TCCT~T~T~C~TGTAT~CC~~~TC~C~~C~CTCTCGA~ SEARYTDIILYVHGSLALVLLLLLAGVYHRQAIHGHHSRQ250 L R L RG A S A v P +I340 t700

.

.

.

.

.

E

R

+360 .

+300

.

.

A

A

.

P

P

.

GCCTGTCACTGTACAWLAGCTGTCCCffiTTCCCTTTffiC~~GTTCT~TT~GTC~~TCCTCT~GT~GTTTGTCCCT~T~~~TGTCC~TCTCCTC~GT~

LVRGVRLSSSGZ90 S G C960 +920 . . . . . . . . CCW;CCCTTGCTCACGGGCCTTGTWLGTCTAGACCTACCTCTC~TC~CTTT~GTTCCCCC~~~TffiTGCTC~GCCCCT~T~~T~TTT~~GTffiT PPLLTGLVSLDLPLDPLWEFPRDRLVLGKPLGEGCFGQVV330 P;TVQKLSRFPLARQFSLESRSSGKSSLS

A

.

A

.

.

+1020

.

.

.

+1oao

.

.

TTGTGCAGAAGCCCTTGGCATGGATTCCTCCCGGCCAGAC-CCA~TCGT~TGT~~T~T~~~T~TCC~C~~ATTT~~CCT~TCTC~A~T~ CAEALGMDSSRPDQTSIVAVKMLKDNASDKDLADLISEME370 A T P A F P t1200 t1140

.

.

.

.

GATGATGAAGCTAATCGGAAGACATT~CCT~TG~TGTC~CTCA~~CCC~TATGTGATTGT~TATGC~~ MMKLIGRHKNIINLLGVCTQEGPLYVIVEYAAKGNLREFL410

.

. V

. . GXAAACCTTCGGGAATTCCT

C

V

+1320

t1260 . . . . CAGGCCCTGATCTCAGCCCTGATGGGCCTCGWLGCAGCGA CCGTGUXGGCGTCCCC RARRPPGPDLSPDGPRSSEGPLSFPALVSCAYQVARGMQY450

.

V +I380 . . . TCTGGAGTCTCGGAAGTGCRTCCACCGGGACCTGGCTGCC LESRKCIHRDLAARNVLVTEDDVMKIADFGLARGVHHiDY490

.

.

.

.

.

.

.

.

.

.

t1440 .

.

t1560

.

.

CTATAAGAAAACCAGCAATGGCCGCCTGCCAGTCAAGTM;T YKKTSNGRLPVKWMAPEALFDRVYTHQSDVWSFGILLWEI530 t1620 . . . . . TCW%ATACCCCGGCATCCCAGTGWLGGAGCn;TTCTCACTT CTTCACCCTCGGGGGC FTLGGSPYPGIPVEELFSLLREGHRMERPPNCPSELYGLM570

+16SO .

D +1740 . . . . . CTTTTAAGCAGCTGGTGGAAGCTCTGWLCAAGGTCCTGCT GAC&GAGTGTTGGCACGCACXTCCTTCTCAGAGGCCGA RECWHAAPSQRPTFKQLVEALDKVLLAVSEEYLDLRLTFG610

w +1eoo

+I860 +1920 . . . . . ACCCTATTCCCCCAACAATGT~~G~~ffiT~TCCTC~~CTCGGTTTT~GC~C~~CTTT~CC~~~~~C~TT~~TTTCCT~~A~C~C PYSPNNGDASSTCSSSDSVFSHDPLPLEPSPFPFPEAQTT650 SC OS 8 +19ao 1-2040 . . . . ~~CTGGGAACWLTGTTGCATGGGCTCGTA~CCGT~CGT~CTC~CCTGTTT~TCA~TTTGACGTT~CTGT~T~~CTCT~CTC~~TACT~T~

P

.

.

GSGVQ

l

t2100 t2160 . . . . . . . CCAGATCCTCTCTCTGGCCCTGTTTT~A~C~T~TTffiTCTT~TT~~GTT~~CTTCTGTTC-CTTATGTTC~~T~~GTT~CTCCTCGTCT~~T +2220 +22ao . . . . . . CATGGTCGTGCCCTTGGACTCATCCTCCT-~~~TT~~CTT~~CTTA~CT~CCC~~TCTC~CTGACT~TCTTT~TCCT~~TTTCTA~ +2340 t2400 . . . . . . . TCCCCAAACAACCTAGAGGCCTCGGGACTTCACTGULCCCCC~CCC~~C~~~CTC~CAC~T~TCCCC~CTCCC~CT~TTGTTCTA~TCTTGTT~~~CT t2460 +2520 . . . CAGCTCTGGTGTCCTTGAGAGAGGGAAGCCTGTGGAAAAW

. . .

293 EXPERIMENTAL

AND DISCUSSION

of r-FGFR4 cDNA Degenerate oligo primers were designed according to a reverse translation of the published aa sequence of the chicken fig FGFR subtype (Lee et al., 1989). Adult rat lung had been previously observed by 1251-autoradiography to contain very high concentrations of bFGF binding (William Herblin, unpublished observations). Therefore, mRNA from rat lung was used as the starting material for the synthesis of oligo(dT)-primed cDNA. One of the eleven pairs of degenerate oligo primers tested was able to amplify a 770-bp band that was subsequently cloned and sequenced. The computer-generated translation of the nt sequence ORF revealed several regions of aa homology to the chicken Jig sequence. Nondegenerate oligo primers were then made according to the sequence information obtained from the PCR-amplified band and were used to amplify the 5’ and 3’ ends of the cDNA using the RACE technique (Frohman et al., 1988). Three overlapping fragments representing the 5’, middle, and 3’ portions of the FGFR4 cDNA were thus obtained. At least two independent isolates were cloned and sequenced for each of these three fragments to control for the possibility of PCR-induced mutations. The three overlapping cDNA fragments were spliced into one contiguous fragment using the technique of PCRmediated assembly of overlapping DNA fragments (Ho et al., 1989; Horton et al., 1989) to yield a final assembled 2.7-kb cDNA. The first ATG codon appears at nt 92 and is followed by an ORF that encodes a 660-aa protein (Fig. 1) and ends with a TGA stop codon. The deduced protein contains a hydrophobic region of 21 aa (Ile228 to Tyr248) which resembles a transmembrane domain. While this work was in progress, the sequence of the human FGFR4 (BFGFR4) cDNA was published (Partanen et al., 1991). The nt and deduced aa sequences of h-FGFRI are 86% and 92% identical, respectively, to the sequences shown in Fig. 1. On the basis of this close ho-

(a) Cloning and characterization

kb 9.5 7.5

28s

-

4.4--

2.4 18s

-

i::

-

Fig. 2. Northern

blot analysis

of various

Total RNA (10 ng) was isolated Sprague-Dawley

rats) and resolved

rose gel (Sambrook position

of 28s and 18s ribosomal

sulfate/40%

Ficoll, and 20 pg of sheared out overnight

salmon

formamide/

Madison,

represent

WI) consisting

nylon

membranes

at 42°C for 5 h

serum

albumin/0.04%

sperm DNA per ml. Hybridization

in the same solution containing

1.5 x IO5 cpm/

System kit, Promega,

of the 5’ terminal 721 nt of the r-FGFR4

twice for 5 min at room temperature after a 2-week exposure

clone.

in 2 x SSCjO. 1%

SDS and twice for 30 min at 55°C in 0.1 x SSC/O.l% iogram was developed

agakb) and

x SSC/7 mM Tris pH

bovine

ml of a 32P-labeled DNA fragment (Nick Translation Blot was washed

Methods.

(300 g male

are labeled at the left side of

to GeneScreenm

polyvinyl-pyrrolidone/O.O4%

was carried

(numbers

and the blot was prehybridized

in 40 ml of 10% dextran 7.6/0.04x

RNAs

were transferred

Nuclear),

rat tissues

on a 2.2 M formaldehyde-1.2%

et al., 1989). Size markers

the figure. RNAs (New England

rat tissue mRNAs.

from various

SDS. Autorad-

at -80°C.

mology, the lung isolate appears to be the rat homologue of the h-FGFRI sequence. However, the cDNA sequence described here does not encode a hydrophobic signal se-

Fig. 1. The nt sequence of the rat lung cDNA isolate, r-FGFR4, and the deduced aa sequence. Numbers and dots above every line refer to nt numbers; aa are aligned with the lirst nt of each codon, and numbers are marked at the end of each line. The putative ATG start and stop codons are underlined. The postulated

transmembrane

found in the h-FGFRI sequence.

domain

is shown with double underlining.

gene that differ from the rat sequence.

Methods. Rat lung poly(A)+mRNA

was reverse transcribed

cDNA was made from 0.2 pg methyl mercury-denatured passed

over a Linkers

5 column

(5 Prime-3

gene (Lee et al., 1989) was performed (the coding potential

for aa EMEMMKMI,

represent

letters shown below the rat aa sequence aa residues

from Invitrogen.

using the oligo TTCTCGAGAATTCTAGA(T,,).

Prime), diluted to 500 ~1, and boiled for 5 min. A reverse translation programs

(containing

(Devereux

for aa DPQPHIQW)

the residues

relative to the rat

Briefly, single-stranded

The cDNA-RNA of the aa sequence

et al., 1984) and was used to design degenerate

the coding potential

represent

that are deleted from the human

using the Red Module kita and protocol

poly(A)+RNA

using the Wisconsin

primers 5’-GAYCCNCARCCNCAYATHCARTGG

The boldfaced

The A symbols

hybrid was

of the chicken Jig

oligo primers.

The oligo

and 5’-ATCATYTTCATCAMCCAMC

where Y = C or T; R = A or G; N = A, C, G or T; and H = A,C or T) were used to amplify the initial 770-

bp receptor fragment. PCR reactions were generally carried out at 94°C for 1 min, primer annealing temperature for 1 mm, and 72°C for 3 min for 29 cycles followed by a 7 min chase at 72°C. The primer annealing temperature was calculated as described by Sambrook et al. (1989). An annealing temperature of 55°C was used for the degenerate oligos. All sequencing reactions were performed using Sequenase Version 2.0 sequencing kit from US Biochemical (Cleveland, OH) following the protocol provided. The 5’ and 3’ ends of the rat FGFR4 cDNA were amplified essentially as described by Frohman

et al. (1988). This sequence

has been deposited

in the GenBank

database

under accession

No. M91599.

294 quence, the acidic box characteristic of the FGFR members, and the first Ig domain, as described for the human version (Partanen et al., 1991.) (b) Tissue-specific expression of r-FGFR4 The expression of the r-FGFR4 gene was determined using Northern blot analysis of the total RNA isolated from various rat tissues. Using the region of the cDNA encompassing the putative extracellular domain (nt -50 to + 671 as shown in Fig. 1) as a hybridization probe, a RNA species of approximately 3.0-3.2 kb was detected in the lung and kidney (Fig. 2). The identity of the faintly hybridizing band at approx. 4.2 kb observed in the spleen is unknown but may be due to an alternatively spliced version of the r-FGFR4 mRNA, use of an alternative poly(A)’ signal, or possibly cross-hybridization to another related FGFR subtype. No hybridizing mRNA species were detectable in any of the other tissues analyzed: liver, brain, testes, heart, fat pads, stomach, or intestines. The tissue-specific pattern of expression observed in Fig. 2 differs considerably from the pattern shown by Partanen et al. (1991). These authors observed expression of FGFR4 in many tissues including the liver, spleen, and intestine which are not detectable on the autoradiogram shown in Fig. 2. This might be explained, however, by the fact that the Northern blot mRNA samples used by Partanen et al. (1991) were obtained from 17-18-week-old human fetuses, whereas the samples used in Fig. 2 were obtained from adult rat tissues and organs.

(3) Intriguingly, analogous forms of cDNAs encoding the Jig (termed the ‘ y N-terminal motif in Hou et al., 1991) and the bek (A. Yayon, personal communication) FGF

receptor subtypes have been observed in which initiation presumably begins at the first Met encountered, 16 aa Nterminal to the Cys residue that delineates the beginning of the second Ig domain (Met’ in Fig. 1). Like the y N-terminal motif of Hou et al. (1991), the isolate described in this work may therefore represent a potential intracellular form of the FGFR4 receptor. Studies are now underway to investigate further the subcellular localization of FGFR4 in the lung and kidney to test this hypothesis.

ACKNOWLEDGEMENTS

We wish to thank Donna Pedicord for her kind gift of the rat tissue RNAs and Northern blot used in the tissue distribution analysis, and William Herblin, Janet L. Dzubow, and Kristie Eidsvoog for helpful discussions, encouragement, and impetus for carrying out these studies.

REFERENCES Devereux,

J., Haeberh,

quence analysis

0.: A comprehensive

set of se-

for the VAX. Nucleic Acids Res. 12 (1984)

387-395. Dionne, CA.,

(c) Conclusions It should be noted that there is an inconsistency between the size of the cloned r-FGFR4 cDNA (2.7 kb) and the size of the transcript (3.0 kb) as detected in adult rat lung by Northern blot analysis. There are several possible explanations for this. (I) No consensus polyadenylation recognition sequence (Sheets et al., 1990) is discernible at the 3’ end of the cDNA. Since the 3’ untranslated region becomes very dA + dT-rich toward the C terminus, it is likely that the d(T)i7 oligo used to prime the rat lung poly(A)+ RNA annealed to and primed from a stretch of A’s in this region. Priming from an internal string of A’s could have caused a truncation of the 3’ untranslated region that would account for at least some of the observed difference in size between the cDNA and mRNA. (2) A deletion at or truncation from the 5’ end of the cDNA could also be a contributing factor since the r-FGFR4 appears to be truncated by 144 aa in comparison with the three Ig domain form of the h-FGFR4 aa sequence. However, several independent isolates of the rFGFR4 cDNA were obtained by RACE technology (each reaction used a fresh aliquot of cDNA), and all clones began with the same C- 91 (Fig. 1).

P. and Smithies,

programs

Crumley,

M., Burgess, pression

G., Bellot, F., Kaplow,

J.M., Searfoss,

W.H., Jaye, M. and Schlessinger,

of two distinct

high-alhnity

acidic and basic fibroblast

receptors

growth factors.

G., Ruta,

J.: Cloning

and ex-

cross-reacting

with

EMBO J. 9 (1990) 2685-

2692. Frohman,

M.A.,

full-length

Dush,

cDNAs

gene-specific

M.K.

and Martin,

G.R.:

from rare transcripts:

oligonucleotide

primer.

Rapid

production

amplification

of

using a single

Proc. Natl. Acad.

Sci. USA 85

(1988) 8998-9002. Goldfarb,

M.: The fibroblast

growth factor family. Cell Growth

Differ. 1

(1990) 439-445. Ho,

S.N.,

Hunt,

H.D.,

Site-directed chain reaction. Horton,

R.M.,

Engineering

Horton,

mutagenesis

R.M.,

Pullen,

by overlap

J.K.

extension

and Pease,

L.R.:

using the polymerase

Gene 77 (1989) 51-59.

Hunt,

H.D.,

Ho,

S.N.,

Pullen,

J.K.

and Pease,

hybrid genes without the use of restriction

splicing by overlap

extension.

Hou, J., Kan, M., McKeehan,

L.R.:

enzymes: gene

Gene 77 (1989) 61-68. K., McBride,

G., Adams,

P. and McK-

eehan, W.L.: Fibroblast growth factor receptors from liver vary in three structural domains. Science 251 (1991) 665-668. Houssaint,

E., Blanquet,

riglia, A., Courtois, factor

receptor

P.R., Champion-Amaud, Y. and Breathnach,

genes in the human

P., Gesnel, M.C., Tor-

R.: Related fibroblast

genome.

growth

Proc. Natl. Acad.

Sci.

USA 87 (1990) 8180-8184. Isacchi,

A., Bergonzoni,

human receptor

L. and Sarmientos,

P.: Complete

for acidic and basic fibroblast

Acids Res. 18 (1990) 1906. Johnson, D.E., Lee, P.L., Lu, J. and Williams, receptor for acidic and basic fibroblast 10 (1990) 4728-4736.

sequence

growth factors.

of a

Nucleic

L.T.: Diverse forms of a

growth factors.

Mol. Cell. Biol.

295 Johnson,

D.E.,

human

arrangement

H., Werner,

S. and Williams,

L.T.: The

growth

growth factor receptor

genes: a common

structural

10 (1991) 1347-1354.

Lu, J., Chen,

fibroblast

underlies

the mechanisms

for generating

that differ in their third immunoglobulin

domain.

receptor

forms

Mol. Cell. Biol. 11

K., Johnson,

of an additional ily, FGFR-3.

member of the fibroblast Proc. Natl. Acad.

Lee, P.L., Johnson, Purification fibroblast Mansukhani,

D.E., Williams, L.T., and Hayman,

D.E., Cousens, factor.

CHO cells is activated

for basic

D., Talarico, growth

D., Levytska,

factor

by basic FGF

(FGF)

receptor

and Kaposi

FGF.

and Aaronson,

receptor

by creation

S.A.: Expression of a transforming

expressed

in

Proc. Natl.

Claesson-Welsh,

T.P.,

Eerola,

EMBO

protein-tyrosine

J.

kinase

E.B. and Singer, S.J.: Identilication

ulated protein-tyrosine

of a developmentally

kinase by using anti-phosphotyrosine

ies to screen a cDNA

expression

reg-

antibod-

library. Proc. Natl. Acad. Sci. USA

Sambrook,

J., Fritsch,

Laboratory

E.F.

Manual,

Sheets,

M.D.,

AAUAAA

Ogg,

and Maniatis,

T.: Molecular

2nd ed. Cold Spring Harbor

Cloning.

Laboratory

A

Press,

NY, 1989.

S.C.

and

Wickens,

and the poly(A)-addition

M.P.:

Point

mutations

site: effects on the accuracy

efficiency of cleavage and polyadenylation

in and

in vitro. Nucleic Acids Res.

18 (1990) 5799-5805. cDNA cloning of the KGF autocrine

loop. Proc.

Natl.

Sci. USA 89 (1992) 246-250. J., Make&

family of embryonic

pattern.

Proc. Natl. Aca. Sci. USA 87 (1990) 5812-5816.

Cold Spring Harbor, V. and Basilica,

D.P., Fleming, T.P., Smith, CL., Burgess, W.H., Chan,

A.M.-L. Acad.

L.S., Fried, V.A. and Williams, L.T.: DNA cloning of a receptor

expression

86 (1989) 5449-5453.

Sci. USA 87 (1990) 4378-4382.

Miki, T., Bottaro,

Partanen,

fam-

Science 245 (1989) 57-60.

A., Moscatelli,

C.: A murine fibroblast Acad.

growth factor receptor

Sci. USA 88 (1991) 1095-1099.

and complementary growth

M.J.: Isolation

with a distinct

E.B.: A distinctive

receptors. Pasquale,

(1991) 4627-4634. Keegan,

Pasqurde,

factor receptor

S., Duan, D.-S.R.,

Williams,

L.T.:

fibroblast

growth

with different

E., Korhonen,

L. and Alitalo, K.: FGFR4,

Werner,

J., Hirvonen,

H.,

a novel acidic tibroblast

82-88.

de Vries, C., Peters, K.G., Johnson,

Differential factor

ligand-binding

splicing receptor

in the extracellular 1 generates

specificities.

receptor

D.E. and region

of

variants

Mol. Cell. Biol. 12 (1992)